Field of the Invention
The field of the invention relates generally to optical microscopy systems based on multi-photon photoacoustic imaging. More particularly, the present disclosure is directed to optical microscopy systems that use modulation techniques and contrast agents to enable the systems to extract nonlinear photoacoustic signals for imaging with high spectrum sensitivity and frequency selectivity.
Description of Prior Art
Photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) are emerging technologies allowing optical absorption contrast and ultrasonic resolution to be combined in one single modality to achieve high resolution imaging at a penetration depth that is beyond optical mean free path. However, the focus of the present disclosure is on PAM, rather than PACT.
PAM can be categorized into acoustic resolution PAM (AR-PAM) and optical resolution PAM. (OR-PAM). AR-PAM weakly focuses a pulsed laser beam onto a sample to generate ultrasound by local thermal expansion, and the spatial resolution is around tens of micrometers which is within the millimeters penetration depth but beyond the existing depth limit of optical imaging modalities. The lateral resolution of AR-PAM depends on the center frequency and the numerical aperture (NA) of the ultrasonic transducer, whereas the axial resolution depends on the bandwidth of the ultrasonic transducer. High ultrasonic frequency leads to high spatial resolution. Since the acoustic attenuation coefficient is proportional to the ultrasonic frequency, the higher the ultrasonic frequency is the lower the ultrasonic penetration limit is. Generally speaking, the upper limit of the ultrasonic frequency is around 300 MHz.
To further enhance the lateral resolution of PAM, OR-PAM, which employs fine optical focusing, is proposed as an alternative. While the optical lateral resolution of OR-PAM is enhanced because of the confined photoacoustic excitation, the axial resolution is still derived primarily from the time-resolved ultrasound detection. As a result, the penetration depth of OR-PAM is only comparable to that of a conventional high-resolution optical imaging modality.
Both AR-PAM and OR-PAM heretofore focus on the single-photon excited photoacoustic effect which induces acoustic signals along the entire light path within the sample. These signals can only be ultrasonically time-resolved. The spatial resolution of AR-PAM is defined mainly by ultrasonic parameters. The lateral and axial resolution of OR-PAM are defined by optical and ultrasonic parameters, respectively. If the center frequency of the transducer is, for example, within 1 to 100 MHz, the corresponding optimal spatial resolution is limited to approximately 1.5 millimeters to 15 micrometers, and the corresponding imaging depth is limited to a few centimeters to tens of micrometers into biological tissues. It is infeasible to try to use the conventional PAM to attain imaging with spatial (i.e., both axial and lateral) resolution that is beyond the ultrasonic wavelength range when the penetration depth is limited to within millimeters.
In order to overcome the physical limitations of the conventional PAM, multi-photon photoacoustic microscopy (MPPAM), a hybrid technique combining multi-photon absorption and PAM, is recently proposed. According to nonlinear optics, multi-photon absorption is a special nonlinearity observed typically when local photon density is extremely high. For example, by focusing a pulsed laser into a sample, the high light intensity in the center of the focal area induces nonlinearity. Nonlinear microscopy such as two-photon absorption fluorescent microscopy takes advantage of this optical phenomenon to achieve sub-micron spatial resolution while using infrared photons to suppress attenuation of the tissue.
Similarly, taking advantage of the photoacoustic effect induced by multi-photon, MPPAM is capable of fine optical sectioning because the generated photoacoustic signals are well confined in the objective focal area. The spatial resolution of MPPAM almost solely depends on the dimensions of the objective focal volume. Having pure optical characteristics, MPPAM provides optical resolution rather than ultrasonic resolution and the imaging depth is within optical diffusion limit.
To induce multi-photon photoacoustic effect, it requires not only high instantaneous excitation of optical power but also efficient energy transformation in a sample from absorbed photons to phonons. Since strong single-photon absorption usually dominates the overall energy transformation process, the generated nonlinear signals are often buried in noises. Therefore, to induce multi-photon photoacoustic effect, there are two critical barriers must be overcome, detection of the weak nonlinear signals and complete separation of nonlinear signals from linear ones.
To implement MPPAM, a method used currently is to use a nanosecond laser with low repetition rate (<10 kHz) and high pulse energy (mJ/pulse) to excite the sample, and then analyze ultrasonic signals in time domain in the presence of a band-pass filtering element which increases the signal-to-noise ratio (SNR). However, several issues make this method infeasible for biomedical imaging. First, the high pulse energy causes photo-toxicity and damages to the tissue of an organism. Second, because light pulse with low repetition rate generates wide-band stimulation in frequency domain, the detection in time domain is unable to provide the spectral sensitivity and selectivity as required. Third, when linear absorption and nonlinear absorption coexist in the tissue, they tend to mix up and become indistinguishable from each other in the absence of modulation. Therefore, in order to attain high resolution and deep tissue imaging as desired, a new method which can overcome these obstacles is needed.
Prior art literatures are disclosed and discussed as follows.
Literature 1: “Thermoacoustic Microscopy” (Allen Rosencwaig et al., U.S. Pat. No. 4,255,971, 1981). This patent is the earliest patent relating to photoacoustic effect that mentions modulation. It provides an abstract concept of various modulation methods without explaining how to implement the concept. The pulsed laser, as the main excitation source of the invention, is hardly mentioned in the patent. Furthermore, it neither discusses multi-photon photoacoustic effect nor does it apply contrast agents to improve the image contrast ratio. The patent touches on an abstract concept without describing practical applications or implementations.
Literature 2: “Ultrafast Measurement of Two-photon Absorption by Loss Modulation” (P. Tian et al., Opt. Lett. Vol. 27, No. 18, 2002). This article discloses a loss modulation technique to precisely measure the efficiency of two-photon absorption.
Literature 3: “High-resolution Confocal Microscopy by Saturated Excitation of Fluorescence” (Ki. Fujiida et al., Phys. Rev. Lett. 99, 228105, 2007). This article discloses the application of the loss modulation technique in fluorescent microscopy and multi-photon fluorescent microscopy. Although the manner the signal is processed and the contrast is generated are similar to that of the present disclosure, it is different from the present invention because it acquires the image through fluorescent light detection rather than ultrasonic detection.
Literature 4: “Fine Depth Resolution of Two-photon Absorption-induced Photoacoustic Microscopy using low-frequency bandpass filtering” (Yoshihisa Yamaoka et al., Opt. Express, Vol. 19, No. 14, 2011); Literature 5: “Frequency-selective Multi-photon-excitation-induced Photoacoustic Microscopy (MEPAM) to Visualize the Cross Sections of Dense Object” (Yoshihisa Yamaoka et al., Proc. Of SPIE, Vol. 7564, 2010); and Literature 6: “Enhancement of Multi-photon-excitation-induced Photoacoustic Signals by Using Gold Nanoparticles Surrounded by Fluorescent Dyes” (Yoshihisa Yamaoka et al., Proc. Of SPIE, Vol. 7177, 2009).
Literature 4-6 describes the multi-photon-excitation-induced photoacoustic effect and the mainstream detection methods at the time. Yet, these detection methods, such as the wideband detection and the time domain analysis, cannot achieve spectral sensitivity and selectivity as desired. These methods are inherently different from the present disclosure which uses narrow-band detection and frequency domain analysis.
Literature 7: “Non-resonant Multi-photon Photoacoustic Spectroscopy for Noninvasive Subsurface Chemical Diagnostics” (Nirmala Chandrasekharan et al., Applied Spectroscopy, Vol. 58, 2004). This article discloses a novel spectroscopy based on multi-photon absorption photoacoustic effect. In this study, a nanosecond pulsed laser light is focused on a sample to induce nonlinear acoustic signal, and an unfocused ultrasonic transducer is used for detection. Moreover, the spectral absorption of exogenous absorber such as Rhodamine and tryptophan and endogenous ones inside tumors are measured and analyzed. Despite the similarity in the generation of multi-photon photoacoustic effect to MPPAM, the applications and original purposes of the spectroscopy are distinct from the microscopy of the present disclosure. Furthermore, this study uses time domain analysis with broadband detection and utilizes no modulation schemes. It indicates that there is no exact mechanism to distinguish the linear signal from the nonlinear signal, which is a core issue of MPPAM. Therefore the scope of this study is completely different from the present disclosure.
Literature 8: “Ultrahigh Resolution Photoacoustic Microscopy via Transient Absorption” (Ryan L. Shelton et. al., Biomed. Opt. Express 1. No. 2, 2010). This article discloses a square wave modulation on a light source. The system uses two modulators in the chopping mode to modulate a pump beam and a probe beam, respectively. It then uses an objective lens to focus the two beams into a sample, and measures the transient response of the induced photoacoustic signal by an ultrasonic probe. The system also integrates the signal with respect to the difference frequency and sum frequency. However, the manner of its modulation is different from that of the present disclosure, which uses pure sinusoidal modulation. In addition, the signal extraction method of this study is a pump-probe technique, instead of a loss-modulation technique. Finally, this study fails to address the problems incurred during the square wave modulation, the induced even harmonics in particular when the modulator operates in the non-sinusoidal modulation mode.